Trans zeatin

Solanum lycopersicum L. cv. Micro-Tom was used in this study. Tomato plants were grown in a growth chamber under conditions of 21–25°C with 16 h light at 4000–6000 l×/8 h dark. CRISPR/Cas9 vectors were transformed into Agrobacterium tumefaciens strain GV2260 and introduced into tomato cotyledons by the leaf disk method according to a previous study with slight modification (Sun et al., 2006 (link)). Sterilized tomato seeds were germinated on MS medium and cotyledons (5–7 days after germination) were cut into small pieces of approximately 0.5–0.7 cm and then transformed with Agrobacterium (OD₆₀₀ = 0.01) in 40 ml infection medium [1X MS liquid medium (pH5.7), 1.2 μl 2-mercaptoethanol (Sigma-Aldrich), 100 μM acetosyringone (TCI chemicals)]. The explants were transferred to MS medium containing 40 μM acetosyringone and cultured in the dark for 3 days in a growth chamber, then transferred onto MS-agar medium containing 100 mg/L kanamycin, 1.0 mg/L trans-zeatin (Wako), and 25 mg/L meropenem (Wako). Four weeks after transformation, transgenic calli were selected using kanamycin and GFP selection as a Cas9 expression marker (Ueta et al., 2017 (link)). GFP positive calli were cut using a scalpel under a fluorescence stereoscopic microscope M165FC (Leica) for use in further mutation analysis.

The chemicals used in this study were obtained from the following providers: DMBQ (Sigma−Aldrich, St Louis, MO, USA), acetosyringone (Tokyo Chemical Industry Co., Tokyo, Japan), syringic acid (Sigma−Aldrich), kinetin (Sigma−Aldrich), 2-isopentenyladenine (2iP), tetrafluoro-1,4-benzoquinone (TFBQ, Sigma−Aldrich), alpha-bromoacetosyringone (ASBr, Toronto Research Chemicals, North York, Canada), 6-benzylaminopurine (BA, Wako Pure Chemical Co., Osaka, Japan), trans-zeatin (Wako Pure Chemical Co.) and thidiazuron (Wako Pure Chemical Co.). LGR-991 (Nisler et al., 2010 (link)) was a kind gift from Dr. Lukáš Spíchal at the Czech Advanced Technology and Research Institute.